The primary reason for conducting this study was to discover whether AZT-based HIV-1 prophylaxis was significantly associated with the induction of mtDNA mutations in uninfected infants born to HIV-1-infected mothers. Answers to this question were obtained in a stepwise fashion. First, we determined that a previously reported horizontal DGGE method [Michikawa et al., 1997
] could be adapted to a vertical DGGE procedure for comprehensive, rapid, and sensitive detection of rare to common mutations/polymorphisms occurring in the tRNA genes and flanking regions of the human mitochondrial genome. Second, analysis of umbilical cord tissue using vertical DGGE revealed modest numbers of mainly synonymous polymorphisms in unexposed newborns compared to increased levels of nonsynonymous sequence variants at both polymorphic and novel sites in AZT-exposed infants. Third, estimates of the mutant fractions for each mtDNA change showed a range in the degree of heteroplasmy among sequence variants from AZT-exposed infants versus mostly homoplasmic polymorphisms in unexposed controls. Last, classification of the sequence variants based upon reports in the literature or the nature of the mutations suggested that several mtDNA changes were potentially pathogenic.
Umbilical cord tissue was selected for analysis because it is accessible and isolated mitochondria originate primarily from energy-demanding endothelium and smooth muscle cells of the umbilical vein and two umbilical arteries. The remaining structural elements of the umbilical cord are arranged to support these blood vessels crucial to the developing fetus. Very few mitochondria occur in the enveloping amnion epithelial cell layer and mucous connective tissue called Wharton's jelly, that consists of relatively few stellate cells, collagen fibers, and abundant extracellular matrix of various glycosaminoglycans and cavernous spaces that contribute to the elasticity of the umbilical cord [Takechi et al., 1993
; Eyden et al., 1994
; Sexton et al., 1996
; Sobolewski et al., 1997
]. Thus, mtDNA alterations found in umbilical cord tissue may reflect events occurring in vascular cells throughout the body.
DGGE or related techniques, such as denaturing HPLC or temporal temperature gradient gel electrophoresis (TTGE), have significant advantages over other methods used for mtDNA mutation detection in that it permits the simultaneous detection of more than one mutation occurring in an amplified region of DNA, it can detect several types of mutations including any base substitution, frame-shift, or small deletion/insertion, and it can provide limited information about the mutant fraction for a mtDNA sequence variant [Myers et al., 1985
; Lerman et al., 1987; Walker and Skopek, 1993
; Michikawa et al., 1997
]. Other methods, including Southern blotting, long-extension PCR, single-stranded conformation polymorphism (SSCP), PCR-restriction fragment length polymorphism analysis, and solid-phase mini-sequencing, using PCR with allele-specific oligonucleotides, are limited in the scope of mutations that can be identified using a single approach [Kajander et al., 1999
; Boles et al., 2003
]. The current study used DGGE to screen for sequence variants in ~21% of the mitochondrial genome of umbilical cord tissue from AZT-exposed versus healthy unexposed infants. As discussed below, however, two research groups have previously employed sensitive methods for genome-wide analysis of small-scale mtDNA mutations in PBMCs of NRTI-treated HIV-1-infected adult patients.
McComsey et al. 
investigated the molecular mechanisms of NRTI-associated mitochondrial dysfunction by, in part, using TTGE and DNA-sequencing analysis to screen for mtDNA sequence variants in PBMCs from 10 NRTI-treated HIV-1-infected patients, four ARV-naïve HIV-1-infected individuals, and 10 healthy adult controls. More than 20 different base substitutions (including changes at polymorphic sites and novel mutations) were found in NRTI-naïve and NRTI-treated patients; most of these sequence variants denoted benign homoplasmic polymorphisms. The authors suggested that NRTI-induced mutations are tissue-specific or that NRTIs may unmask pre-existing mtDNA variations in HIV-1 disease and promote clinical mitochondrial dysfunction. A follow-up study was conducted to assess the utility of serial blood samples in characterizing the relationships between HIV-1 infection, NRTI therapies, acquisition of mtDNA mutations, and clinical mitochondrial toxicity [McComsey et al., 2005
]. The study population included NRTI-treated HIV-1-infected adults (n
= 54), ARV-naïve HIV-1-infected patients (n
= 33), and healthy adult controls (n
= 48) followed for up to 52-months. The report only included mtDNA mutation data for the two HIV-1-infected patients who showed detectable differences in TTGE banding patterns over time in serial blood samples. Both patients (one drug-naïve subject given d4T plus emtricitabine and one drug-experienced patient receiving AZT-3TC) had one or more sequence variants that were heteroplasmic in the first blood specimen but homoplasmic after months of NRTI treatment, suggesting a dynamic process where NRTI therapy appears associated with acquisition, segregation, and expansion of these mtDNA changes. The authors believed that NRTI-treated patients might have additional variants at a low level of heteroplasmy undetectable by their analytical methods.
Martin et al. 
used SSCP and DNA-sequencing analysis to investigate the impact of NRTI therapy on the development, frequency, and nature of mtDNA mutations in PBMCs prior to (T1 samples) and after 6–77 months of treatment (T2 samples). Their study population consisted of 16 drug-naïve HIV-1-infected patients who were placed on NRTI treatment and a reference group of 10 drug-naïve, untreated HIV-1-infected subjects [Martin et al., 2003
]. Based upon T1 and T2 samples from these 26 patients, five NRTI-treated subjects incurred increased numbers of novel heteroplasmic mutations over time. Four of five patients with mtDNA mutations developed evidence of peripheral fat wasting (lipoathrophy) between blood sample intervals (P
= 0.031), suggestive of a pathogenic potential for NRTI-related mtDNA mutations. There also was a trend toward more nonsynonymous base substitutions in T2 samples than T1 samples, with the numbers of synonymous substitutions being similar in the before and after NRTI-treatment samples. The authors concluded that NRTI therapy provides conditions permissive for the emergence over time of “random” mtDNA mutations; however, they could not cite evidence for positive selection of pre-existing non-wild-type sequences.
The finding of seemingly “random” mtDNA mutations in earlier studies of HIV-1-infected adults may be partly a consequence of the variety of NRTI treatment protocols used. In the reports of McComsey et al. [2002
, ARV-experienced HIV-1-infected patients, as a group, received more than a dozen different triple-drug therapies including one or two NRTIs. In the work of Martin et al. 
, NRTI-treated patients were given one of three different NRTI drug combinations, including d4T-3TC (n
= 10), AZT-3TC (n
= 3), or d4T-didanosine (n
= 3) [with sequence variants detected in 3, 1, and 1 subject(s) in their respective groups]. In these earlier studies, the finding of mutations formed in a “random” fashion would be highly likely in that different individual NRTIs, or pairs of NRTIs, should not cause the same specific mtDNA mutations. Rather, individual NRTIs, or combinations of NRTIs, possess different mutagenic potencies and specificities in human lymphoblastoid cells [Carter et al., 2007
]. Therefore, it is not surprising to find no overlap in the sequence variants occurring in AZT-3TC exposed newborns in the current work () versus those detected in earlier studies of NRTI-treated HIV-1-infected adults. It is also noteworthy that adults have slower rates of cell replication than developing fetuses, possibly leading to the finding of greater numbers of sequence variants at birth. These dissimilarities in sequence variants found in PBMCs versus umbilical cord tissue of NRTI-exposed subjects are unlikely due to tissue-specific differences because preliminary data from our group suggests that AZT-exposed newborns have similar mtDNA mutations in umbilical cord tissue (current study) and cord blood lymphocytes (unpublished data). On the other hand, the finding of two AZT-3TC exposed infants with the same novel mtDNA mutation (, PID #s 231 and 234) is suggestive of targeted chemical mutagenesis at a potential mutational hot spot.
In our study, we cannot establish whether the increased detection of sequence changes at two polymorphic sites in AZT-exposed infants (i.e., 9-base deletion at nucleotide 8271 and A10398G transition, ) resulted from the unmasking of silent sequence variants or chemical induction of mutations at predisposed sites in mtDNA. However, given that the 9-base pair deletion at nucleotide 8271 is typically a polymorphism associated with Asian, North American Indian, and Polynesian populations [Hertzberg et al., 1989
; Harihara et al., 1992
; Lorenz and Smith, 1994
], or isolated cases in European or Latin populations [Torroni et al., 1995
], the occurrence of this deletion in one Hispanic unexposed infant versus four Hispanic AZT-exposed infants () raises the question as to whether these variants are rare preexisting polymorphisms that have been unmasked or are caused by NRTI treatment.
Among the NRTIs used as ARVs, AZT has clearly been shown to be genotoxic in standard in vitro and in vivo assays [IARC, 2000
; Walker and Poirier, 2007
; Wogan, 2007
], and mounting evidence suggests that it also triggers mitochondrial damage and mutation [Poirier et al., 2004
; Walker et al., 2004
; Kohler and Lewis, 2007
]. In infants receiving prepartum AZT-3TC, a significant increase in the frequency of HPRT
reporter gene mutations was driven by an increase in transversion mutations [O'Neill et al. 2001
]. Likewise, mutations found in the K-ras
cancer genes of lung neoplasms from mice exposed transplacentally to AZT were predominantly transversions [Hong et al., 2007
]. In contrast, 11/12 heteroplasmic base substitutions observed in AZT-3TC exposed infants in the current study involved transition mutations, leaving open the question of the mechanism(s) leading to the formation of these mtDNA mutations. Lim and Copeland 
have shown that AZT-monophosphate (at clinically relevant levels) inhibits the exonuclease activity of DNA pol γ, contributing to an increase in spontaneous mutations which are preferentially transitions. These authors further found that AZT-related mitochondrial toxicity in vivo may result, in part, from moderately efficient incorporation and very inefficient removal of AZT from mtDNA, yielding an increase in mutations. AZT has also been shown to inhibit TK2
, the mitochondrial targeted thymidine kinase, which could cause a decrease or alteration in the dNTP precursor pools needed for mtDNA replication [Lynx and McKee, 2006
; Susan-Resiga et al., 2007
]. Imbalanced mitochondrial dNTP pools have been shown to alter the fidelity of DNA pol γ [Song et al., 2005
]. Other mechanisms underlying the toxicities of AZT have been proposed, but additional work is needed to gain a better understanding of the mutagenic modes of action of AZT and their long-term consequences.
For ethical reasons, the current study did not include a treatment-control group of drug-naïve infants born to untreated HIV-1-infected mothers to assess the mutagenic potential of the fetal environment in the presence of maternal HIV-1 infection. In the United States, women are routinely treated with ARVs upon diagnosis of HIV-1 infection, making it problematic to address the degree to which fetal responses to “HIV-1 exposure,” in the absence of maternal ARV treatment, contributes to perinatal toxicities and long-term health risks in the offspring [Poirier et al., 2003
; Funk et al., 2007
]. Consequently, there is limited evidence that fetal responses and/or “HIV-1 exposure” damage mitochondria of fetal cells/tissues, perhaps increasing the risk for mtDNA mutations. In collaboration with the Womens and Infants Transmission Study (WITS), Poirier et al. 
found significant mtDNA depletion in leukocytes of uninfected infants born to HIV-1-infected mothers who received no treatment, with this depletion being further increased in infants of mothers receiving AZT during pregnancy. These findings suggest that factors related to maternal HIV-1 infection can affect the integrity of mitochondria and possibly play an undefined role in the perturbations in genetic markers observed in uninfected children. In the future, it is important to test the hypothesis that fetal environment in the face of maternal HIV-1 infection and absence of NRTI treatment has mutagenic potential leading to changes in nDNA and mtDNA that may be preventable in uninfected infants [Funk et al., 2007
The findings related to clinical mitochondrial dysfunction in uninfected children receiving perinatal NRTIs have been the subject of considerable debate as illustrated by the recent opinion piece [Blanche et al., 2006
] and editorial response [Spector and Saitoh, 2006
] published in the journal AIDS
. Interim reports of cord blood leukocyte mtDNA depletion and umbilical cord mitochondrial morphological damage and depletion indicated that most clinically asymptomatic infants exposed to NRTIs in utero do have molecular evidence of mitochondrial compromise in the absence of clinical manifestations of mitochondrial disease [Benhammou et al., 2007
]. In the current study, the discovery of increases in mtDNA sequence variants in umbilical cord tissue of uninfected newborns receiving prepartum AZT-3TC is troublesome because umbilical vessel endothelium and smooth muscle cells are the predominant source of mtDNA, and the same cell types occur throughout the body including mitochondrial-rich tissues such as the heart. Initial reports of Lipshultz et al. [2000
suggested that perinatal exposure to AZT monotherapy was not associated with cardiac abnormalities in uninfected children. More recent preliminary findings in children receiving multiple ARV drug therapies suggest different risks. The 2- to 3-year follow-up of larger numbers of HIV-1-uninfected ARV-exposed infants suggest that these children have significantly less ventricular mass and wall thickness compared to HIV-1-uninfected children who were not exposed in utero to ARV-based therapy. These differences appear to persist for 2–3 years after birth, mandating long-term cardiac outcome studies [Lipshultz, 2008
; Lipshultz et al., 2005
]. As NRTI-exposed children age, long-term studies also are needed to delineate the expression and clinical consequences of multiple induced mtDNA mutations compared to single pathogenic mutations arising from maternal inheritance. Foster and Lyall 
argue that the cumulative NRTI-associated toxicities are becoming progressively apparent in the clinic, and, in an era of expanding treatment options, understanding and minimizing toxicities becomes a possibility.